Composition for dermal injection

ABSTRACT

The present invention relates to a composition for dermal injection which includes two or more types of cross-linked hyaluronic acid particles having different particle diameters and non-cross-linked hyaluronic acid. The composition for dermal injection according to the present invention satisfies viscosity, extrusion force, and viscoelasticity conditions for dermal injection, and an extrusion force deviation is low so that the user does not feel fatigue when the composition is injected into the dermal thereof. Also, the composition is excellent in viscoelasticity and tissue restoring ability, is maintained for a long period of time, allows rapid recovery because an initial swelling degree is low, and also is excellent in safety and stability in the body.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No.PCT/KR2018/002400 having International filing date of Feb. 27, 2018,which claims the benefit of priority of Korean Patent Application No.10-2017-0026490 filed on Feb. 28, 2017. The contents of the aboveapplications are all incorporated by reference as if fully set forthherein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a composition for dermal injection.

Hyaluronic acid, as a gel type product having transparency andviscosity, is a biodegradable and highly hydrophilic substance, andplays an important role in maintaining moisture in the dermal, dermalvolume, and dermal elasticity because it attracts 214 water moleculesper one molecule thereof. Thus, a filler containing hyaluronic acid asan ingredient has been used for restoration of facial dermal elasticity,subtle improvement of a contour, reduction of facial wrinkles, andgeneral cosmetic facial contouring procedures.

However, since natural hyaluronic acid has a half-life of only 1 to 2days, the hyaluronic acid used in the filler is made in a cross-linkingstate to be maintained in the dermal for a long period of time. Here,the crosslinking results in preventing the degradation of hyaluronicacid caused by hyaluronidases and increasing viscosity to form volume(Song, Yi-Seop et al., Korean Journal of Dermatology 2014;52(2):100˜105).

Hyaluronic acid fillers currently available on the market are in themonophasic or biphasic form. A monophasic filler is composed of ahomogeneous gel so that it has high viscosity, is smoothly injected, andis useful for forming a delicate shape. A biphasic filler is made in theform of a particle by filtering a gel using a sieve so that it has highelasticity, and thus it is possible to maintain shape and increasevolume.

Meanwhile, research on the development of a filler having ideal in vivocharacteristics and surgical usefulness is continuing. However, ahyaluronic acid filler having excellent in vivo stability has high gelhardness and high viscosity so that it may be difficult to inject thefiller through a fine gauge needle. Also, a hyaluronic acid fillercapable of being easily injected through a fine gauge needle may havelow in vivo stability. Accordingly, hyaluronic acid fillers excellent inboth viscosity and elasticity are required.

SUMMARY OF THE INVENTION

The present invention is directed to providing a composition for dermalinjection which includes two or more types of cross-linked hyaluronicacid particles having different particle diameters and non-cross-linkedhyaluronic acid.

The present invention provides a composition for dermal injection whichincludes first cross-linked hyaluronic acid particles, secondcross-linked hyaluronic acid particles having different particlediameters from the first cross-linked hyaluronic acid particles, andnon-cross-linked hyaluronic acid, wherein the first cross-linkedhyaluronic acid particles and the second cross-linked hyaluronic acidparticles are included in a weight ratio of 1:2.5 or more to 5.5 orless, and the first cross-linked hyaluronic acid particles and thenon-cross-linked hyaluronic acid are included in a weight ratio of 1:0.1or more to 1.2 or less.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1, FIG. 2, FIG. 3, FIG. 4 and FIG. 5 are graphs illustratingextrusion force deviations of hyaluronic acid compositions according toPreparation Examples 1-6, 1-7, and 1-11 to 1-13.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention relates to a composition for dermal injectionwhich includes first cross-linked hyaluronic acid particles, secondcross-linked hyaluronic acid particles having different particlediameters from the first cross-linked hyaluronic acid particles, andnon-cross-linked hyaluronic acid.

Hereinafter, the configurations of the present invention will bedescribed in detail.

In the present invention, a composition for dermal injection may bedenoted as a filler composition.

In the present invention, numerical values are presented usingexpressions such as “A or more”, “A or less”, “more than A” and “lessthan A”, but in the case of numerical values given without such anexpression, it is to be understood that the meaning of “A or more” or “Aor less” is implied.

In the composition for dermal injection according to the presentinvention, the first cross-linked hyaluronic acid particles and thesecond cross-linked hyaluronic acid particles may be included in aweight ratio of 1:2.5 or more to 5.5 or less, specifically 1:2.5 or moreto 3.5 or less.

In addition, the first cross-linked hyaluronic acid particles and thenon-cross-linked hyaluronic acid may be included in a weight ratio of1:0.1 or more to 1.2 or less, specifically 1:0.1 or more to less than0.5.

Within the above ranges, properties required for the composition fordermal injection, such as viscosity, extrusion force, viscoelasticity,and the like, may be achieved.

Hyaluronic acid is a linear polymer including β-D-N-acetylglucosamineand β-D-glucuronic acid alternately bonded to each other, and may beinterpreted as including all of hyaluronic acid itself, a salt thereof,and a combination thereof in the present invention. The hyaluronic acidmay have a molecular weight of 100,000 to 5,000,000 Da or 1,000,000 to1,500,000 Da, but the present invention is not limited thereto. Examplesof the salt of hyaluronic acid include inorganic salts such as sodiumhyaluronate, potassium hyaluronate, calcium hyaluronate, magnesiumhyaluronate, zinc hyaluronate, cobalt hyaluronate, and the like andorganic salts such as tetrabutylammonium hyaluronate and the like. Inthe present invention, as the hyaluronic acid, hyaluronic acid itselfand a salt thereof may be used alone or in combination of two or more.The hyaluronic acid or the salt thereof may be isolated from amicroorganism, synthesized, or commercially available, but the presentinvention is not limited thereto. For example, the hyaluronic acid maybe isolated from Streptococcus sp. (Streptococcus equi or Streptococcuszooepidemicus) and purified.

In the present invention, the cross-linked hyaluronic acid particles maybe used in the same sense as hydrated cross-linked hyaluronic acidparticles. For example, it may mean that hyaluronic acid has beensubjected to a crosslinking reaction through a covalent bond using ahydroxyl group. The moisture content or crosslinking ratio of hyaluronicacid may be adjusted through a common method used in the related art,and may be, for example, 10 to 20 mol % or 10 to 15 mol %.

The hyaluronic acid particles may be crosslinked by a crosslinkingagent. The crosslinking agent may be, but is not limited to, ethyleneglycol diglycidyl ether (EGDGE), 1,4-butanediol diglycidyl ether (BDDE),1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether,polypropylene glycol diglycidyl ether, diglycerol polyglycidyl ether,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), divinyl sulfone(DVS), biscarbodiimide (BCDI), or a combination thereof.

The composition for dermal injection according to the present invention(hereinafter, also referred to as a hyaluronic acid composition)includes two or more types of cross-linked hyaluronic acid particleshaving different particle diameters.

In the present invention, the first cross-linked hyaluronic acidparticle has lower elasticity and higher cohesion than those of thesecond cross-linked hyaluronic acid particle. For example, at afrequency of 0.01 Hz to 1 Hz, the first cross-linked hyaluronic acidparticle may exhibit a G′ value of less than 300 Pa and a tan δ value of0.3 or more.

The tan δ value is a G″/G′ value (damping factor), which is a numericalvalue indicating whether the material is close to a solid or liquidstate. Here, G′ represents elastic modulus, and G″ represents viscousmodulus. A tan δ value close to 1 at a frequency of 0.01 to 1 Hz mayrepresent a solution state (low elasticity), and a tan δ value close to0 may represent an elastic body with high elasticity. Also, it has beenreported that as a tan δ value is low and the percentage of elasticity(100×G′/(G′+G″)) is high, the duration of a filler is expected to belong.

The first cross-linked hyaluronic acid particles may have an averageparticle diameter of 10 to 250 μm, specifically, 20 to 200 μm, 50 to 150μm, 80 to 130 μm, 20 to 100 μm, 100 to 200 μm, 200 to 250 μm, 50 to 100μm, or 150 to 200 μm.

In the present invention, the average particle diameter is D50 (50%diameter of particle), which means a particle size (volume) of aparticle corresponding to the 50 percentile in the particle sizedistribution curve. Such an average particle diameter is measured usinga particle size analyzer (Malvern, MS3000), and water is used as adispersing solvent. That is, the average particle diameter represents aparticle diameter of hydrated cross-linked hyaluronic acid particles.

In the present invention, properties of the second cross-linkedhyaluronic acid particle are adjusted according to the size thereof. Thesecond cross-linked hyaluronic acid particle has low viscosity andexcellent elasticity compared to those of the first cross-linkedhyaluronic acid particle. For example, at a frequency of 0.01 to 1 Hz,the second cross-linked hyaluronic acid particle may exhibit a G′ valueof 300 Pa or more and a tan δ value of less than 0.3.

The second cross-linked hyaluronic acid may have an average particlediameter of 1,500 to 3,000 μm, specifically 1,800 to 2,800 μm, 2,000 to2,700 μm, 2,300 to 2,500 μm, 1,500 to 2,000 μm, 2,000 to 2,500 μm, 2,500to 3,000 μm, 1,700 to 2,200 μm, 2,200 to 3,000 μm, 1,500 to 2,500 μm, or2,000 to 3,000 μm.

The composition for dermal injection according to the present inventionincludes non-cross-linked hyaluronic acid. The non-cross-linkedhyaluronic acid is in the form of a solution and may impart fluidity tothe composition for dermal injection.

In the present invention, the first cross-linked hyaluronic acidparticles, second cross-linked hyaluronic acid particles, andnon-cross-linked hyaluronic acid may be included at 1 to 10 parts byweight with respect to 100 parts by weight of the entire composition.

The composition for dermal injection according to the present inventionmay further include epidermal growth factor (EGF) in addition to theabove-described components. The EGF may be injected into the dermal tostimulate the production of collagen, a fibroblast, and elastin, forexample, to increase an effect of tissue restoration.

The EGF may be included at 0.0001 to 0.002 part by weight with respectto 100 parts by weight of the entire composition.

In addition, the composition for dermal injection according to thepresent invention may further include an anesthetic component. Theanesthetic may alleviate pain experienced during injection of thecomposition.

Such an anesthetic component may be, but is not limited to, ambucaine,amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine,bupivacaine, butacaine, butamben, butanilicaine, butethamine,butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon,dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine,beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,zolamine, or a combination thereof.

The anesthetic component may be included at 0.1 to 1 part by weight withrespect to 100 parts by weight of the composition.

The composition according to the present invention may be used with theaddition of other common additives such as an antioxidant, a buffersolution and/or a bacteriostat, a diluent, a dispersant, a surfactant, abinder, a lubricant, or the like as necessary.

The composition for dermal injection according to the present inventionmay have the following physicochemical properties.

(a) no bubbles in appearance, colorlessness, and transparency;

(b) a pH of 7±1;

(c) a viscosity of 65,000 to 95,000 cP;

(d) an average osmotic pressure of 0.325 osmol/kg±10%;

(e) an extrusion force of 70 N or less;

(f) an elasticity of 81 to 92%; and

(g) a phase angle of 5 to 15°.

The viscosity is the amount that represents the magnitude of internalfriction in a fluid, which is the resistance of the fluid to flow.Higher viscosity results in ease of injection and is useful for forminga delicate shape. For example, a monophasic hyaluronic acid filler hashigh viscosity so that it is smoothly injected and is useful for forminga delicate shape. In the present invention, viscosity may be measuredusing Brookfield DV3T according to conditions of experimental examplesto be described below.

The composition for dermal injection according to the present inventionmay have a viscosity of 65,000 to 95,000 cP, 70,000 to 90,000 cP, or73,000 to 82,000 cP. The extrusion force means extrusion force at aninjection rate at which a patient feels comfortable. The expression “apatient feels comfortable” is used to define an injection rate whichdoes not cause injuries or excessive pain in a patient when thecomposition is injected into the dermal. The term “comfort” used hereinencompasses the comfort or ability of a doctor or medical professionalto inject the composition as well as the comfort of a patient. In thepresent invention, extrusion force may be measured using T0-101-161commercially available from TestOne Co., Ltd. according to conditions ofexperimental examples to be described below. Generally, when extrusionforce is low, there is no pressure pain during injection of thecomposition, and injection may be easily controlled.

The composition for dermal injection according to the present inventionmay have an extrusion force of 70 N or less or 65 N or less.

Viscoelasticity is the property of a material that exhibits both liquidand solid characteristics when a force is applied to the material. Inthe present invention, viscoelasticity may be measured using a rheometeraccording to conditions of experimental examples to be described below.Specifically, a force resisting an applied force and a loss of energy inthe composition due to the applied force may be measured using arheometer to determine viscous modulus, elastic modulus, and phaseangle.

Elastic modulus (storage modulus; G′) means the ratio of strain andstress which an elastic body has within the elastic limit. As theelastic modulus is higher, a composition is rigid and has a greaterability to resist strain.

The composition for dermal injection according to the present inventionmay have an elastic modulus of 800 to 1,200 or 850 to 1,000.

Viscous modulus (loss modulus; G″) is a measure of lost energy andpertains to a viscous component of a material.

The composition for dermal injection according to the present inventionmay have a viscous modulus of 100 to 200 or 100 to 130.

In addition, elasticity may be calculated by the following formula usingelastic modulus and viscous modulus values. As the elasticity is higher,tissue restoring ability is excellent, and the duration of a filler isprolonged.Elasticity (%)=(100×G′/(G′+G″))

The composition for dermal injection according to the present inventionmay have an elasticity of 81 to 92% or 85 to 90%.

In addition, the phase angle is a measure of whether the composition isclose to a liquid or solid state. As the phase angle is lower, thecomposition has solid characteristics, and as the phase angle is higher,the composition has liquid characteristics. In the case of a high phaseangle, when deformation occurs due to a force applied from the outsideor facial expression, the recovery to the original state is delayed, andthe original shape is not maintained. In the case of a low phase angle,the G″ value becomes smaller due to an instantaneous response toexternal deformation factors so that the composition is more like anelastic body than a fluid, and thus the composition does not flow out,and the original shape thereof may be continuously maintained. Thus, itis very important to appropriately maintain elasticity and phase anglevalues in the composition for dermal injection.

The composition for dermal injection according to the present inventionmay have a phase angle of 5 to 13° or 5 to 10°.

The composition for dermal injection according to the present inventionmay be prepared through a method commonly used in the related art.

In addition, the present invention provides a method of restoringtissue, which includes administering the above-described composition fordermal injection to a mammal.

The mammal may be a human.

The tissue restoration refers to temporarily or semi-permanentlyalleviating body wrinkles or restoring a wrinkle-free state, improvingcontours, forming volume in the tissue, or regenerating tissues such asin scar healing by injecting the composition. The dermal and tissuerefer to those in the face, breast, hip, sexual organ, and other bodyregions.

In particular, the composition for dermal injection according to thepresent invention may be selected appropriately according to the degreeof wrinkling of a user graded in accordance with the WSRS standard. TheWSRS is an acronym for Wrinkle Severity Rating Scale, and classifies thedegree of wrinkling of a human into 5 grades (Grades 1 to 5). The Grades1 to 5 are absence (no of folds), mild (shallow folds), moderate(moderate folds), severe (deep folds), and extreme (very deep folds),respectively. Detailed contents of the WSRS and each grade are describedin a document by Am J Clin Dermatol 2004; 5 (1): 49-52 1175-0561, andthe present invention can evaluate wrinkles in accordance with the WSRSusing a method presented in the document.

Meanwhile, the Ministry of Food and Drug Safety in Korea also classifiesthe wrinkling degree into mild, moderate, severe, and extreme accordingto the WSRS through guidelines for approval and review of a dermalcosmetic filler based on a hyaluronic acid raw material issued onDecember 2017, which proposes to include information on wrinkling degreein describing the purpose of a dermal cosmetic filler.

The composition according to the present invention may be used forsevere folds of Grade 4 or extreme folds of Grade 5 in the WSRS.

In addition, a syringe may be filled with the composition for dermalinjection to inject the composition into the layers of dermal.

The layers of dermal are classified into the epidermis, dermis, andhypodermis. The composition for dermal injection according to thepresent invention may be injected into the deep dermis or subdermis.

Hereinafter, the present invention will be described in more detail withreference to embodiment examples of the present invention. However, thefollowing examples are merely presented to exemplify the presentinvention, and the content of the present invention is not limited tothe following examples. That is, the examples of the present inventionserve to complete the disclosure of the present invention, and areprovided to make known the full scope of the invention to those ofordinary knowledge and skill in the art to which this inventionpertains. This invention should be defined based on the scope of theappended claims.

EXAMPLES Reference Example. Measurement of Properties

(1) Viscosity Measurement

The viscosity was measured according to a viscosity measurement methodamong the general test methods of the Korean Pharmacopoeia.

Specifically, 500 ul of a composition sample was loaded in a sample cupof a viscometer (DV3T, Brookfield), the sample cup was installed in aCP-52 sample cup, and the rotational speed of the spindle was then setto 2 rpm to measure viscosity.

(2) Extrusion Force Measurement

A compression test was performed using a universal testing machine(T0-101-161, TestOne).

Specifically, a syringe was filled with a composition sample, a 27G½-inch needle was installed to the syringe, and the syringe was then setin a jig. Afterward, a speed of 50 mm/min and a displacement of 25 mmwere set to perform a compression test.

(3) Viscoelasticity Measurement

Rheological properties were measured using a rheometer.

Specifically, a sample was placed between parallel plates, a forceresistant to an applied force and a loss of energy were measured whilevibrating and rotating the parallel plates to determine the elasticmodulus (G′), viscous modulus (G″), and phase angle of the sample.

Conditions for rheometer analysis are as follows.

Frequency: 1 Hz

Temperature: 25° C.

Strain: 5%

Measuring geometry: 20 mm plate

Measuring gap: 0.5 mm

Measuring mode: oscillation mode

In addition, elasticity was calculated by the following formula withreference to the measured G′ and G″ values.Elasticity (%)=(100×G′/(G′+G″))

Preparation Example. Preparation of Composition for Dermal Injection

(1) Preparation of First Cross-Linked Hyaluronic Acid Particles

10 g of sodium hyaluronate, 81 g of purified water, and 9 g of 1 Msodium hydroxide (1M NaOH) were stirred at 400 rpm under vacuum untilthe mixture became a transparent gel without granules. Then, 0.5 g ofbutanediol diglycidyl ether (BDDE) as a crosslinking agent was addedthereto and stirred. After the stirring was completed, the container wassealed and a crosslinking reaction was performed under conditions of 80rpm and 50° C. for 1 hour. Then, a resulting substance was allowed tostand at 27° C. for 16 hours to prepare a gel.

Afterward, the gel thus obtained was input into 30 L of a 0.9× phosphatebuffered saline (PBS) solution, and then the PBS solution was exchangedwith a new one every 3 hours (3 times/day for 5 days) to eliminate aresidual reagent. Then, a resulting substance was passed through amortar grinder (RS 200 commercially available from Retsch GmbH) for 40minutes to prepare first cross-linked hyaluronic acid particles.

The first cross-linked hyaluronic acid particles thus prepared had anaverage particle diameter of about 200 μm.

(2) Preparation of Second Cross-Linked Hyaluronic Acid Particles

20 g of sodium hyaluronate, 117 g of purified water, and 13 g of 1M NaOHwere stirred at 400 rpm under vacuum until the mixture became atransparent gel without granules. Then, 1 g of BDDE as a crosslinkingagent was added thereto and stirred. After the stirring was completed,the container was sealed and a crosslinking reaction was performed underconditions of 80 rpm and 50° C. for 1 hour. Then, a resulting substancewas allowed to stand at 27° C. for 16 hours to prepare a gel.

Afterward, the gel thus obtained was input into 30 L of a 0.9×PBSsolution, and then the PBS solution was exchanged with a new one every 3hours (3 times/day for 5 days) to eliminate a residual reagent. Then, aresulting substance was passed through a 1000 μm standard test sieve toprepare second cross-linked hyaluronic acid particles.

The second cross-linked hyaluronic acid particles thus prepared had anaverage particle diameter of about 2,000 μm.

(3) Preparation of Non-Cross-Linked Hyaluronic Acid

2 g of sodium hyaluronate was added to 100 g of purified water andstirred to prepare 2% non-cross-linked hyaluronic acid.

(4) Preparation of Hyaluronic Acid Composition

The first cross-linked hyaluronic acid particles prepared in step (1),the second cross-linked hyaluronic acid particles prepared in step (2),and the non-cross-linked hyaluronic acid were mixed in predeterminedcontents (g) and content ratios to prepare a hyaluronic acidcomposition.

Example 1. Evaluation of Properties of Hyaluronic Acid CompositionIncluding Only One or Two of First Cross-Linked Hyaluronic AcidParticle, Second Cross-Linked Hyaluronic Acid Particle, andNon-Cross-Linked Hyaluronic Acid

(1) Preparation of Hyaluronic Acid Composition (Comparative PreparationExamples 2-1 to 2-16)

As a comparative example of the composition including all of firstcross-linked hyaluronic acid particles, second cross-linked hyaluronicacid particles, and non-cross-linked hyaluronic acid, hyaluronic acidcompositions were prepared in the same manner as the preparation exampleexcept that the first cross-linked hyaluronic acid particles, secondcross-linked hyaluronic acid particles, and non-cross-linked hyaluronicacid were mixed in contents (g) and content ratios as shown in thefollowing Table 1.

TABLE 1 First Second cross-linked cross-linked hyaluronic hyaluronicNon-cross-linked acid particle acid particle hyaluronic acid ContentContent Content Content ratio Content ratio Content ratio Comparative 101 0 0 0 0 Preparation Example 2-1 Comparative 10 1 10 1 0 0 PreparationExample 2-2 Comparative 10 1 20 2 0 0 Preparation Example 2-3Comparative 10 1 30 3 0 0 Preparation Example 2-4 Comparative 10 1 40 40 0 Preparation Example 2-5 Comparative 10 1 50 5 0 0 PreparationExample 2-6 Comparative 0 0 10 1 0 0 Preparation Example 2-7 Comparative0 0 10 1 2 0.2 Preparation Example 2-8 Comparative 0 0 10 1 4 0.4Preparation Example 2-9 Comparative 0 0 10 1 6 0.6 Preparation Example2-10 Comparative 0 0 0 0 1 1 Preparation Example 2-11 Comparative 10 1 00 2 0.2 Preparation Example 2-12 Comparative 10 1 0 0 4 0.4 PreparationExample 2-13 Comparative 10 1 0 0 6 0.6 Preparation Example 2-14Comparative 10 1 0 0 8 0.8 Preparation Example 2-15 Comparative 10 1 0 010 1.0 Preparation Example 2-16

(2) Measurement of Properties

Measurement results of viscosity, extrusion force, and viscoelasticityof each of the hyaluronic acid compositions according to ComparativePreparation Examples 2-1 to 2-16 were shown in the following Tables 2and 3.

TABLE 2 First Second Non- cross-linked cross-linked cross-linkedExtrusion hyaluronic hyaluronic hyaluronic Viscosity force acid particleacid particle acid (cP) (N) Comparative 1 0 0 25896 31.12 PreparationExample 2-1 Comparative 1 1 0 75694 87.08 Preparation Example 2-2Comparative 1 2 0 108977 123.72 Preparation Example 2-3 Comparative 1 30 111137 144.18 Preparation Example 2-4 Comparative 1 4 0 114169 154.45Preparation Example 2-5 Comparative 1 5 0 125083 163.91 PreparationExample 2-6 Comparative 0 1 0 93950 136.71 Preparation Example 2-7Comparative 0 1 0.2 Unmeasurable Unmeasurable Preparation Example 2-8Comparative 0 1 0.4 Unmeasurable Unmeasurable Preparation Example 2-9Comparative 0 1 0.6 Unmeasurable Unmeasurable Preparation Example 2-10Comparative 0 0 1 16041 11.66 Preparation Example 2-11 Comparative 1 00.2 Unmeasurable Unmeasurable Preparation Example 2-12 Comparative 1 00.4 Unmeasurable Unmeasurable Preparation Example 2-13 Comparative 1 00.6 Unmeasurable Unmeasurable Preparation Example 2-14 Comparative 1 00.8 Unmeasurable Unmeasurable Preparation Example 2-15 Comparative 1 01.0 Unmeasurable Unmeasurable Preparation Example 2-16

As shown in Table 2, the compositions including only first cross-linkedhyaluronic acid particles or only non-cross-linked hyaluronic acidexhibited excessively low viscosity to be inappropriate as a fillercomposition, and the composition including only second cross-linkedhyaluronic acid particles exhibited excessively high extrusion force tobe inappropriate as a filler composition.

In addition, the compositions including first or second cross-linkedhyaluronic acid particles and not including non-cross-linked hyaluronicacid exhibited high viscosity and/or high extrusion force, and thus arenot appropriate for use as a filler composition. In particular, sincenon-cross-linked hyaluronic acid plays a role of lubrication,Comparative Preparation Examples 2-2 to 2-7, which do not includenon-cross-linked hyaluronic acid, may have a problem in that anextrusion force value significantly fluctuates during injection, thereis difficulty in dermal injection, and there is a risk that the user mayfeel pressure pain.

TABLE 3 First Second cross- cross- Non- linked linked cross- hyaluronichyaluronic linked acid acid hyaluronic G′ G″ Phase particles particlesacid Elasticity (Pa) (Pa) angle Comparative 1 0 0 87.80 140.70 19.557.91 Preparation Example 2-1 Comparative 1 1 0 92.17 822.80 69.93 4.86Preparation Example 2-2 Comparative 1 2 0 93.02 1082.00 81.24 4.29Preparation Example 2-3 Comparative 1 3 0 92.88 1246.00 95.54 4.25Preparation Example 2-4 Comparative 1 4 0 93.75 1264.00 84.24 3.81Preparation Example 2-5 Comparative 1 5 0 92.82 1362.00 105.40 3.70Preparation Example 2-6 Comparative 0 1 0 95.97 1433.00 60.16 2.40Preparation Example 2-7 Comparative 0 1 0.2 84.63 1124.00 204.10 10.29Preparation Example 2-8 Comparative 0 1 0.4 77.92 854.70 242.20 15.82Preparation Example 2-9 Comparative 0 1 0.6 70.93 616.00 252.50 22.29Preparation Example 2-10 Comparative 0 0 1 41.01 42.29 60.83 55.19Preparation Example 2-11 Comparative 1 0 0.2 75.77 105.90 33.87 17.73Preparation Example 2-12 Comparative 1 0 0.4 69.44 91.47 40.25 23.75Preparation Example 2-13 Comparative 1 0 0.6 64.45 65.15 35.93 28.88Preparation Example 2-14 Comparative 1 0 0.8 57.82 58.11 42.40 36.11Preparation Example 2-15 Comparative 1 0 1.0 49.34 41.89 43.01 45.76Preparation Example 2-16

In addition, as shown in Table 3, the compositions including first orsecond cross-linked hyaluronic acid particles and not includingnon-cross-linked hyaluronic acid exhibited a low phase angle of 5 orless, and thus are not appropriate as a composition for dermalinjection, and the compositions including first cross-linked hyaluronicacid and non-cross-linked hyaluronic acid exhibited high phase anglevalues, and thus are not appropriate as a composition for dermalinjection.

That is, when all of first cross-linked hyaluronic acid particles,second cross-linked hyaluronic acid particles, and non-cross-linkedhyaluronic acid are included, a hyaluronic acid compound which exhibitsexcellent properties and thus is suitable for dermal injection can beprepared.

Example 2. Evaluation of Properties of Hyaluronic Acid CompositionAccording to Content Ratio of First Cross-Linked Hyaluronic AcidParticles and Second Cross-Linked Hyaluronic Acid Particles

(1) Preparation of Hyaluronic Acid Composition (Preparation Examples 1-5to 1-10 and Comparative Preparation Examples 1-1 to 1-4)

Hyaluronic acid compositions were prepared using first cross-linkedhyaluronic acid particles, second cross-linked hyaluronic acidparticles, and non-cross-linked hyaluronic acid in contents (g) andcontent ratios as shown in the following Table 4.

TABLE 4 First Second cross-linked cross-linked hyaluronic hyaluronicNon-cross-linked acid particle acid particle hyaluronic acid ContentContent Content Content ratio Content ratio Content ratio Comparative 101 10 1 2 0.2 Preparation Example 1-1 Comparative 10 1 10 1 4 0.4Preparation Example 1-2 Comparative 10 1 20 2 2 0.2 Preparation Example1-3 Comparative 10 1 20 2 4 0.4 Preparation Example 1-4 Preparation 10 130 3 2 0.2 Example 1-5 Preparation 10 1 30 3 4 0.4 Example 1-6Preparation 10 1 40 4 2 0.2 Example 1-7 Preparation 10 1 40 4 4 0.4Example 1-8 Preparation 10 1 50 5 2 0.2 Example 1-9 Preparation 10 1 505 4 0.4 Example 1-10

(2) Measurement of Properties

Measurement results of viscosity, extrusion force, and viscoelasticityof each of the hyaluronic acid compositions according to ComparativePreparation Examples 1-1 to 1-4 and Preparation Examples 1-5 to 1-10 areshown in the following Tables 5 and 6.

TABLE 5 First Second Non- cross-linked cross-linked cross-linkedExtrusion hyaluronic hyaluronic hyaluronic Viscosity force acidparticles acid particles acid (cP) (N) Comparative 1 1 0.2 53380 37.71Preparation Example 1-1 Comparative 1 1 0.4 53072 33.16 PreparationExample 1-2 Comparative 1 2 0.2 66996 44.79 Preparation Example 1-3Comparative 1 2 0.4 67955 44.69 Preparation Example 1-4 Preparation 1 30.2 80203 60.04 Example 1-5 Preparation 1 3 0.4 73941 44.19 Example 1-6Preparation 1 4 0.2 90400 66.13 Example 1-7 Preparation 1 4 0.4 8311349.05 Example 1-8 Preparation 1 5 0.2 89287 67.17 Example 1-9Preparation 1 5 0.4 85958 57.15 Example 1-10

As shown in Table 5, the compositions according to ComparativePreparation Examples 1-1 and 1-2, in which a weight ratio of secondcross-linked hyaluronic acid to first cross-linked hyaluronic acid was1:1, exhibited low viscosities of less than 65,000 cP. In the case ofPreparation Examples 1-5 to 1-10, the viscosity satisfied the viscosityrange of 65,000 to 95,000 cP according to the present invention.Particularly, the compositions according to Preparation Examples 1-5 and1-6 exhibited viscosities of 73,000 to 82,000 cP, and thus are the mostappropriate as a filler composition.

Meanwhile, the compositions according to Comparative PreparationExamples 1-3 and 1-4, in which a weight ratio of second cross-linkedhyaluronic acid was 2, exhibited viscosities of 65,000 cP or more, buthad low elasticity as shown in the following viscoelasticity data, andthus are not appropriate as a filler composition.

TABLE 6 First Second cross- cross- Non- linked linked cross- hyaluronichyaluronic linked acid acid hyaluronic Phase particles particles acidElasticity G′ G″ angle Comparative 1 1 0.2 76.73 334.1 101.3 10.74Preparation Example 1-1 Comparative 1 1 0.4 74.64 304.9 103.6 13.40Preparation Example 1-2 Comparative 1 1 0.2 80.36 492.1 120.3 8.28Preparation Example 1-3 Comparative 1 2 0.4 75.52 406.2 131.7 9.70Preparation Example 1-4 Preparation 1 3 0.2 88.84 920.30 115.60 7.16Example 1-5 Preparation 1 3 0.4 87.31 871.10 126.60 8.27 Example 1-6Preparation 1 4 0.2 89.42 1101.00 130.30 6.75 Example 1-7 Preparation 14 0.4 86.36 964.50 152.40 8.98 Example 1-8 Preparation 1 5 0.2 89.53994.90 116.30 6.67 Example 1-9 Preparation 1 5 0.4 87.61 904.40 127.908.05 Example 1-10

In addition, as shown in Table 6, the compositions according toComparative Preparation Examples 1-1 to 1-4, in which a weight ratio ofsecond cross-linked hyaluronic acid to first cross-linked hyaluronicacid was 1 to 2:1, exhibited low elasticity or a high phase angle.

That is, when first cross-linked hyaluronic acid and second cross-linkedhyaluronic acid are included in a weight ratio of 1:2.5 or more to 5.5or less, a hyaluronic acid composition having excellent properties (suchas viscosity, extrusion force, viscoelasticity) can be prepared.

Example 3. Evaluation of Properties of Hyaluronic Acid CompositionAccording to Content of Non-Cross-Linked Hyaluronic Acid

(1) Preparation of Hyaluronic Acid Composition (Preparation Examples1-6, 1-7, and 1-11 to 1-13)

Hyaluronic acid compositions were prepared using first cross-linkedhyaluronic acid particles, second cross-linked hyaluronic acidparticles, and non-cross-linked hyaluronic acid in contents (g) andcontent ratios as shown in the following Table 7.

TABLE 7 First Second cross-linked cross-linked hyaluronic hyaluronicNon-cross-linked acid particle acid particle hyaluronic acid ContentContent Content Content ratio Content ratio Content ratio Preparation 101 30 3 2 0.2 Example 1-6 Preparation 10 1 30 3 4 0.4 Example 1-7Preparation 10 1 30 3 6 0.6 Example 1-11 Preparation 10 1 30 3 8 0.8Example 1-12 Preparation 10 1 30 3 10 1.0 Example 1-13

(2) Measurement of Properties

Measurement results of viscosity, extrusion force, and viscoelasticityof each of the hyaluronic acid compositions according to PreparationExamples 1-6, 1-7, and 1-11 to 1-13 are shown in the following Tables 8and 9.

TABLE 8 First Second Non- cross-linked cross-linked cross-linkedExtrusion hyaluronic hyaluronic hyaluronic Viscosity force acidparticles acid particles acid (cP) (N) Preparation 1 3 0.2 80203 60.04Example 1-6 Preparation 1 3 0.4 73941 44.19 Example 1-7 Preparation 1 30.6 68638 45.20 Example 1-11 Preparation 1 3 0.8 65651 37.97 Example1-12 Preparation 1 3 1.0 68793 32.28 Example 1-13

As shown in Table 8, the viscosities of the hyaluronic acid compositionsaccording to Preparation Examples 1-6, 1-7, and 1-11 to 1-13 satisfiedthe viscosity range of 65,000 to 95,000 cP according to the presentinvention. Particularly, the compositions including non-cross-linkedhyaluronic acid in weight ratios of 0.2 or 0.4 exhibited viscosities of73,000 to 82,000 cP, which are highly appropriate for a filler fordermal injection.

TABLE 9 First Second cross- cross- Non- linked linked cross- hyaluronichyaluronic linked acid acid hyaluronic Phase particles particles acidElasticity G′ G″ angle Preparation 1 3 0.2 88.84 920.30 115.60 7.16Example 1-6 Preparation 1 3 0.4 87.31 871.10 126.60 8.27 Example 1-7Preparation 1 3 0.6 83.96 677.70 129.50 10.82 Example 1-11 Preparation 13 0.8 82.80 695.20 144.40 11.73 Example 1-12 Preparation 1 3 1.0 81.74587.60 131.30 12.59 Example 1-13

In addition, as shown in Table 9, the hyaluronic acid compositionsaccording to Preparation Examples 1-6, 1-7, and 1-11 to 1-13 exhibitedelasticities of 81 to 92 and phase angles of 5 to 13, both of whichsatisfied ranges according to the present invention. Particularly, thecompositions including non-cross-linked hyaluronic acid in weight ratiosof 0.2 or 0.4 exhibited elasticities of 85 to 90 and phase angles of 5to 10, indicating that they have highly appropriate viscoelasticity fora filler for dermal injection.

That is, when first cross-linked hyaluronic acid and second cross-linkedhyaluronic acid are included in a weight ratio of 1:2.5 or more to 3.5or less, and first cross-linked hyaluronic acid and non-cross-linkedhyaluronic acid are included in a weight ratio of 1:0.1 or more to lessthan 0.5, a composition is appropriate for use for dermal injection.

Example 4. Test for Extrusion Force Deviation

For Preparation Examples 1-6, 1-7, and 1-11 to 1-13, the extrusion forcewas measured over time.

The measurement results were plotted on a graph as shown in FIG. 1, FIG.2, FIG. 3, FIG. 4 and FIG. 5.

FIG. 1, FIG. 2, FIG. 3, FIG. 4 and FIG. 5 are graphs illustratingextrusion force deviations of hyaluronic acid compositions according toPreparation Example 1-6, 1-7, and 1-11 to 1-13. It can be confirmed thatwhen all of first cross-linked hyaluronic acid particles, secondcross-linked hyaluronic acid particles, and non-cross-linked hyaluronicacid were included, the extrusion force was about 10 N, and a fillercomposition can be injected uniformly without a large extrusion forcedeviation.

In addition, it can be confirmed that as the content of non-cross-linkedhyaluronic acid increases, the extrusion force deviation becomessmaller.

A composition for dermal injection according to the present inventionsatisfies viscosity, extrusion force, and viscoelasticity conditions fordermal injection, and an extrusion force deviation is low so that theuser does not feel fatigue when the composition is injected into thedermal thereof.

Also, the composition is excellent in viscoelasticity and tissuerestoring ability, is maintained for a long period of time, allows rapidrecovery because an initial swelling degree is low, and also isexcellent in safety and stability in the body.

What is claimed is:
 1. A composition for dermal injection, comprisingfirst cross-linked hyaluronic acid particles, second cross-linkedhyaluronic acid particles having different particle diameters from thefirst cross-linked hyaluronic acid particles, and non-cross-linkedhyaluronic acid, wherein the first cross-linked hyaluronic acidparticles and the second cross-linked hyaluronic acid particles areincluded in a weight ratio of 1:2.5 or more to 5.5 or less, wherein thefirst cross-linked hyaluronic acid particles have an average particlediameter of ≤250 μm and the second cross-linked hyaluronic acidparticles have an average particle diameter of ≥1,500 μm, and the firstcross-linked hyaluronic acid particles and the non-cross-linkedhyaluronic acid are included in a weight ratio of 1:0.1 or more to 1.2or less.
 2. The composition of claim 1, wherein the first cross-linkedhyaluronic acid particles and the second cross-linked hyaluronic acidparticles are included in a weight ratio of 1:2.5 or more to 3.5 orless, and the first cross-linked hyaluronic acid particles and thenon-cross-linked hyaluronic acid are included in a weight ratio of 1:0.1or more to less than 0.5.
 3. The composition of claim 1, wherein thefirst cross-linked hyaluronic acid particles have an average particlediameter of 10 to 250 μm.
 4. The composition of claim 1, wherein thesecond cross-linked hyaluronic acid particles have an average particlediameter of 1,500 to 3,000 μm.
 5. The composition of claim 1, whereinthe first cross-linked hyaluronic acid particle, the second cross-linkedhyaluronic acid particle, or the non-cross-linked hyaluronic acid has amolecular weight of 1,000,000 to 1,500,000 Da.
 6. The composition ofclaim 1, wherein the first cross-linked hyaluronic acid particle or thesecond cross-linked hyaluronic acid particle has a degree ofcrosslinking of 10 to 20 mol %.
 7. The composition of claim 1, whereinthe first cross-linked hyaluronic acid particles, the secondcross-linked hyaluronic acid particles, and the non-cross-linkedhyaluronic acid are included at 1 to 10 parts by weight with respect to100 parts by weight of the entire composition.
 8. The composition ofclaim 1, further comprising epidermal growth factor (EGF).
 9. Thecomposition of claim 8, wherein the epidermal growth factor (EGF) isincluded at 0.0001 to 0.002 part by weight with respect to 100 parts byweight of the entire composition.
 10. The composition of claim 1,further comprising an anesthetic component.
 11. The composition of claim10, wherein the anesthetic component is included at 0.1 to 1 part byweight with respect to 100 parts by weight of the composition.
 12. Thecomposition of claim 1, wherein the composition for dermal injection hasthe following physicochemical properties: (a) no bubbles in appearance,colorlessness, and transparency, (b) a pH of 7±1, (c) a viscosity of65,000 to 95,000 cP, (d) an average osmotic pressure of 0.325osmol/kg±10%, (e) an extrusion force of 70 N or less, (f) an elasticityof 81 to 92%, and (g) a phase angle of 5 to 15°.
 13. The composition ofclaim 12, wherein the composition for dermal injection has a viscosityof 73,000 to 82,000 cP.
 14. The composition of claim 12, wherein thecomposition for dermal injection has an extrusion force of 65 N or less.15. The composition of claim 12, wherein the composition for dermalinjection has an elasticity of 85 to 90% and a phase angle of 5 to 10°.